Single-molecule fluorescence (SMF) enables the detailed study of biological processes with unprecedented resolution. Therefore, SMF has become an important tool for unravelling the biological machinery in cells. SMF has for example been used in examining cellular signal transduction pathways, protein-protein interactions and protein folding. Unfortunately, progress in current SMF technologies may have been hampered by the complex optical set-ups needed to perform such experiments.
Generally, in SMF applications, one wants to probe the fluorescence of single molecules in a large background of other fluorescent molecules, e.g. for studying protein or DNA structure and/or dynamics. This is problematic, as all fluorescent molecules in the illuminated area are probed simultaneously. Since the spot size of a focused laser beam is limited by the diffraction limit, the concentration of fluorescent molecules has to be reduced. Some SMF imaging concepts known in the art may effectively dilute the concentration by randomly switching on and off the molecular fluorescence. However, this is less trivial for probing molecular dynamics, which can be tackled by tightly focusing laser beams in confocal microscopes to reduce the excitation volume and by simultaneously reducing the concentration of fluorescent molecules such that at any time only a few molecules are excited and probed.
Fluorescence is also widely used for sensing of small concentrations of biomolecules in samples, e.g. in body fluids, in order to determine the concentration of certain biomarkers. These sensing principles generally rely on assays with several washing steps to remove dye molecules that have not reacted with analyte molecules. Here as well, a highly confined excitation region provides the possibility to perform wash-free sensing assays, where the binding of the fluorescent molecules on the surface of the substrate can be monitored without the need to wash away the solution after the binding. A wash-free assay allows to measure biomolecular interactions in real-time and greatly simplifies the overall assay protocol and the sample/liquid handling system that is needed. The latter may especially be beneficial for highly integrated, handheld devices.
In the recent past, nanophotonic techniques have been established to reduce the excitation volume. For example, In molecular fluorescence analysis, e.g. of DNA sequences, it is known in the art to immerse a substrate comprising a plurality of pores, e.g. nano-pores, in a liquid comprising the biological sample to analyze and one or more chemically reactive derivatives of at least one fluorophore. For example, the liquid may comprise single stranded DNA and a mixture of the 4 DNA nucleotides, A, C, G and T, each nucleotide labeled with a specific fluorescent tag for emitting a specific colour upon optical excitation. In the pores a processing enzyme, e.g. DNA polymerase, may be immobilized for promoting a chemical reaction between the biological sample to analyze and the chemically reactive fluorophore derivative(s). For example, DNA polymerase in the pore may progressively convert single stranded DNA into double-stranded DNA by adding the complimentary nucleotides one by one. When the biological sample and the chemically reactive fluorophore derivative(s) react under influence of the processing enzyme, e.g. when a nucleotide is incorporated in the DNA strand, it is optically excited by a radiation source that illuminates each pore. After excitation, the fluorescent tag emits radiation of a specific wavelength. A lens may be used to focus the emitted radiation, which may then be recorded by an image sensor. To avoid distortion of the emitted radiation signal, an excitation rejection filter can be used to block excitation radiation from reaching the image sensor, e.g. such that only the emitted radiation reaches the detector. A colour separation element may furthermore be used to spectrally disperse the emitted radiation on different parts of the detector, or on different detectors.
It is furthermore known in the art to surround the pores with a suitable metal, such that the pores may be considered to be zero mode waveguides. The zero mode waveguide (ZMW), e.g. a sub-wavelength aperture in a metal film, can advantageously reduce the excitation volume to the nanometer range. The ZMW approach may for example be particularly suitable for single molecule real-time DNA sequencing.
However, the optics, e.g. lenses, which are used for illumination and collection of the emitted radiation in such prior-art systems can be expensive and bulky. Particularly, expensive microscope setups and high numerical aperture (NA) optics may be required. Furthermore, the colour separation filter needed for dispersing the emitted radiation on different parts of the detector add to the complexity and cost of such system. Another disadvantage of such systems may be that the throughput speed can be rather limited, e.g. the number of pores is limited due to the area constraints imposed by the optics, and a cost-effective parallelization of the technique is hampered by the high cost of the optical components.
While progress has been made on SMF using free-space optics, several bulky optical components and their circuits can also be integrated on chip. The technology of nano-photonics, which is based on high index contrast waveguides, has progressed tremendously and especially optical waveguide circuits based on silicon are becoming a mature technology platform. For example, waveguides constructed from silicon in a SiO2 cladding have shown great potential at telecom wavelengths, although silicon is a strong absorber in the visible wavelength region and therefore not a good material for guiding visible radiation. Molecular fluorescence, however, usually takes place at visible wavelengths, as in this range a lot of high brightness fluorescent dyes are available that can be efficiently coupled to a wide range of biomolecules and cells. In the visible spectrum, alternatives to silicon are, for example, SiN, GaP or TiO2 based waveguides.
For example, the publication “Performance of integrated optical microcavities for refractive index and fluorescence sensing” by Krioukov et al., in Sensors and Actuators B 90, pp. 58-67, a fluorescence sensor is disclosed which comprises integrated waveguides and an integrated optical disk microcavity. Such sensor may obtain a good sensitivity for detecting fluorescence emissions of an analyte. According to this prior disclosure, a microdisk with a radius between 5 and 25 μm and a height between 100 and 255 nm can be excited by a nearby mono-modal straight waveguide via evanescent coupling, while a second waveguide can be used for probing the power inside the microdisk at resonance. An analyte molecule on top of the microdisk will be exposed to an evanescent field which is stronger than the field in an evanescent region of the excitation waveguide. As a result, enhancement in fluorescence emission can be obtained by the factor expressing the power inside the microdisk over the power in the excitation waveguide.
Interactions between optical waveguides and radiation emitters have been explored in the field of integrated photonics. For instance, it has been shown that high index contrast nano-photonic waveguides can excite fluorescence of molecules that reside in the tail of the waveguide mode. On the other hand, photonic crystal cavities have been shown to suppress or enhance luminescence of single radiation emitters using the Purcell effect, e.g. emitters placed in the near field of a resonant cavity emit preferentially in the cavity mode. Generally this effect scales with Q/V, with Q the quality factor of the cavity resonance and V the mode volume. Interestingly, the Purcell effect may act similarly to a filter, with the Q-factor of the resonance determining the rejection rate for non-resonant wavelengths and may promote emission in the desired mode.